The present invention relates to the biopolymer/molecular array scanners and, in particular, to automated adjustment of the focal distance at which a sample biopolymer/molecular array is positioned with respect to laser light sources and photodetectors to produce optimal data acquisition.
The present invention is related to acquisition of biopolymer or molecular-array data and other types of genetic, biochemical, and chemical data from molecular arrays by molecular array scanners. A general background of biopolymer/molecular-array technology is first provided, in this section, to facilitate discussion of the scanning techniques described in following sections.
Array technologies have gained prominence in biological research and are likely to become important and widely used diagnostic tools in the healthcare industry. Currently, molecular-array techniques are most often used to determine the concentrations of particular nucleic-acid polymers in complex sample solutions. Molecular-array-based analytical techniques are not, however, restricted to analysis of nucleic acid solutions, but may be employed to analyze complex solutions of any type of molecule that can be optically or radiometrically scanned and that can bind with high specificity to complementary molecules synthesized within, or bound to, discrete features on the surface of an array. Because arrays are widely used for analysis of nucleic acid samples, the following background information on arrays is introduced in the context of analysis of nucleic acid solutions following a brief background of nucleic acid chemistry.
Deoxyribonucleic acid (xe2x80x9cDNAxe2x80x9d) and ribonucleic acid (xe2x80x9cRNAxe2x80x9d) are linear polymers, each synthesized from four different types of subunit molecules. The subunit molecules for DNA include: (1) deoxy-adenosine, abbreviated xe2x80x9cA,xe2x80x9d a purine nucleoside; (2) deoxy-thymidine, abbreviated xe2x80x9cT,xe2x80x9d a pyrimidine nucleoside; (3) deoxy-cytosine, abbreviated xe2x80x9cC,xe2x80x9d a pyrimidine nucleoside; and (4) deoxy-guanosine, abbreviated xe2x80x9cG,xe2x80x9d a purine nucleoside. The subunit molecules for RNA include: (1) adenosine, abbreviated xe2x80x9cA,xe2x80x9d a purine nucleoside; (2) uracil, abbreviated xe2x80x9cU,xe2x80x9d a pyrimidine nucleoside; (3) cytosine, abbreviated xe2x80x9cC,xe2x80x9d a pyrimidine nucleoside; and (4) guanosine, abbreviated xe2x80x9cG,xe2x80x9d a purine nucleoside. FIG. 1 illustrates a short DNA polymer 100, called an oligomer, composed of the following subunits: (1) deoxy-adenosine 102; (2) deoxy-thymidine 104; (3) deoxy-cytosine 106; and (4) deoxy-guanosine 108. When phosphorylated, subunits of DNA and RNA molecules are called xe2x80x9cnucleotidesxe2x80x9d and are linked together through phosphodiester bonds 110-115 to form DNA and RNA polymers. A linear DNA molecule, such as the oligomer shown in FIG. 1, has a 5xe2x80x2 end 118 and a 3xe2x80x2 end 120. A DNA polymer can be chemically characterized by writing, in sequence from the 5xe2x80x2 end to the 3xe2x80x2 end, the single letter abbreviations for the nucleotide subunits that together compose the DNA polymer. For example, the oligomer 100 shown in FIG. 1 can be chemically represented as xe2x80x9cATCG.xe2x80x9d A DNA nucleotide comprises a purine or pyrimidine base (e.g. adenine 122 of the deoxy-adenylate nucleotide 102), a deoxy-ribose sugar (e.g. deoxy-ribose 124 of the deoxy-adenylate nucleotide 102), and a phosphate group (e.g. phosphate 126) that links one nucleotide to another nucleotide in the DNA polymer. In RNA polymers, the nucleotides contain ribose sugars rather than deoxy-ribose sugars. In ribose, a hydroxyl group takes the place of the 2xe2x80x2 hydrogen 128 in a DNA nucleotide. RNA polymers contain uridine nucleosides rather than the deoxy-thymidine nucleosides contained in DNA. The pyrimidine base uracil lacks a methyl group (130 in FIG. 1) contained in the pyrimidine base thymine of deoxy-thymidine.
The DNA polymers that contain the organization information for living organisms occur in the nuclei of cells in pairs, forming double-stranded DNA helixes. One polymer of the pair is laid out in a 5xe2x80x2 to 3xe2x80x2 direction, and the other polymer of the pair is laid out in a 3xe2x80x2 to 5xe2x80x2 direction. The two DNA polymers in a double-stranded DNA helix are therefore described as being anti-parallel. The two DNA polymers, or strands, within a double-stranded DNA helix are bound to each other through attractive forces including hydrophobic interactions between stacked purine and pyrimidine bases and hydrogen bonding between purine and pyrimidine bases, the attractive forces emphasized by conformational constraints of DNA polymers. Because of a number of chemical and topographic constraints, double-stranded DNA helices are most stable when deoxy-adenylate subunits of one strand hydrogen bond to deoxy-thymidylate subunits of the other strand, and deoxy-guanylate subunits of one strand hydrogen bond to corresponding deoxy-cytidilate subunits of the other strand.
FIGS. 2A-B illustrate the hydrogen bonding between the purine and pyrimidine bases of two anti-parallel DNA strands. FIG. 2A shows hydrogen bonding between adenine and thymine bases of corresponding adenosine and thymidine subunits, and FIG. 2B shows hydrogen bonding between guanine and cytosine bases of corresponding guanosine and cytosine subunits. Note that there are two hydrogen bonds 202 and 203 in the adenine/thymine base pair, and three hydrogen bonds 204-206 in the guanosine/cytosine base pair, as a result of which GC base pairs contribute greater thermodynamic stability to DNA duplexes than AT base pairs. AT and GC base pairs, illustrated in FIGS. 2A-B, are known as Watson-Crick (xe2x80x9cWCxe2x80x9d) base pairs.
Two DNA strands linked together by hydrogen bonds forms the familiar helix structure of a double-stranded DNA helix. FIG. 3 illustrates a short section of a DNA double helix 300 comprising a first strand 302 and a second, anti-parallel strand 304. The ribbon-like strands in FIG. 3 represent the deoxyribose and phosphate backbones of the two anti-parallel strands, with hydrogen-bonding purine and pyrimidine base pairs, such as base pair 306, interconnecting the two strands. Deoxy-guanylate subunits of one strand are generally paired with deoxy-cytidilate subunits from the other strand, and deoxy-thymidilate subunits in one strand are generally paired with deoxy-adenylate subunits from the other strand. However, non-WC base pairings may occur within double-stranded DNA.
Double-stranded DNA may be denatured, or converted into single stranded DNA, by changing the ionic strength of the solution containing the double-stranded DNA or by raising the temperature of the solution. Single-stranded DNA polymers may be renatured, or converted back into DNA duplexes, by reversing the denaturing conditions, for example by lowering the temperature of the solution containing complementary single-stranded DNA polymers. During renaturing or hybridization, complementary bases of anti-parallel DNA strands form WC base pairs in a cooperative fashion, leading to reannealing of the DNA duplex. Strictly A-T and G-C complementarity between anti-parallel polymers leads to the greatest thermodynamic stability, but partial complementarity including non-WC base pairing may also occur to produce relatively stable associations between partially-complementary polymers. In general, the longer the regions of consecutive WC base pairing between two nucleic acid polymers, the greater the stability of hybridization between the two polymers under renaturing conditions.
The ability to denature and renature double-stranded DNA has led to the development of many extremely powerful and discriminating assay technologies for identifying the presence of DNA and RNA polymers having particular base sequences or containing particular base subsequences within complex mixtures of different nucleic acid polymers, other biopolymers, and inorganic and organic chemical compounds. One such methodology is the array-based hybridization assay. FIGS. 4-7 illustrate the principle of the array-based hybridization assay. An array (402 in FIG. 4) comprises a substrate upon which a regular pattern of features are prepared by various manufacturing processes. The array 402 in FIG. 4, and in subsequent FIGS. 5-7, has a grid-like two-dimensional pattern of square features, such as feature 404 shown in the upper left-hand corner of the array. It should be noted that many molecular arrays contain disk-shaped features, rather than round features. Each feature of the array contains a large number of identical oligonucleotides covalently bound to the surface of the feature. These bound oligonucleotides are known as probes. In general, chemically distinct probes are bound to the different features of an array, so that each feature corresponds to a particular nucleotide sequence. In FIGS. 4-6, the principle of array-based hybridization assays is illustrated with respect to the single feature 404 to which a number of identical probes 405-409 are bound. In practice, each feature of the array contains a high density of such probes but, for the sake of clarity, only a subset of these are shown in FIGS. 4-6.
Once an array has been prepared, the array may be exposed to a sample solution of target DNA or RNA molecules (410-413 in FIG. 4) labeled with fluorophores, chemiluminescent compounds, or radioactive atoms 415-418. Labeled target DNA or RNA hybridizes through base pairing interactions to the complementary probe DNA, synthesized on the surface of the array. FIG. 5 shows a number of such target molecules 502-504 hybridized to complementary probes 505-507, which are in turn bound to the surface of the array 402. Targets, such as labeled DNA molecules 508 and 509, that do not contains nucleotide sequences complementary to any of the probes bound to array surface, do not hybridize to generate stable duplexes and, as a result, tend to remain in solution. The sample solution is then rinsed from the surface of the array, washing away any unbound labeled DNA molecules. Finally, the bound labeled DNA molecules are detected via optical or radiometric scanning. FIG. 6 shows labeled target molecules emitting detectable fluorescence, radiation, or other detectable signal. Optical scanning involves exciting labels of bound labeled DNA molecules with electromagnetic radiation of appropriate frequency and detecting fluorescent emissions from the labels, or detecting light emitted from chemiluminescent labels. When radioisotope labels are employed, radiometric scanning can be used to detect the signal emitted from the hybridized features. Additional types of signals are also possible, including electrical signals generated by electrical properties of bound target molecules, magnetic properties of bound target molecules, and other such physical properties of bound target molecules that can produce a detectable signal. Optical, radiometric, or other types of scanning produce an analog or digital representation of the array as shown in FIG. 7, with features to which labeled target molecules are hybridized similar to 706 optically or digitally differentiated from those features to which no labeled DNA molecules are bound. In other words, the analog or digital representation of a scanned array displays positive signals for features to which labeled DNA molecules are hybridized and displays negative features to which no, or an undetectably small number of, labeled DNA molecules are bound. Features displaying positive signals in the analog or digital representation indicate the presence of DNA molecules with complementary nucleotide sequences in the original sample solution. Moreover, the signal intensity produced by a feature is generally related to the amount of labeled DNA bound to the feature, in turn related to the concentration, in the sample to which the array was exposed, of labeled DNA complementary to the oligonucleotide within the feature.
Array-based hybridization techniques allow extremely complex solutions of DNA molecules to be analyzed in a single experiment. An array may contain from hundreds to tens of thousands of different oligonucleotide probes, allowing for the detection of a subset of complementary sequences from a complex pool of different target DNA or RNA polymers. In order to perform different sets of hybridization analyses, arrays containing different sets of bound oligonucleotides are manufactured by any of a number of complex manufacturing techniques. These techniques generally involve synthesizing the oligonucleotides within corresponding features of the array through a series of complex iterative synthetic steps, or depositing oligonucleotides isolated from biological material.
As pointed out above, array-based assays can involve other types of biopolymers, synthetic polymers, and other types of chemical entities. For example, one might attach protein antibodies to features of the array that would bind to soluble labeled antigens in a sample solution. Many other types of chemical assays may be facilitated by array technologies. For example, polysaccharides, glycoproteins, synthetic copolymers, including block copolymers, biopolymer-like polymers with synthetic or derivitized monomers or monomer linkages, and many other types of chemical or biochemical entities may serve as probe and target molecules for array-based analysis. A fundamental principle upon which arrays are based is that of specific recognition, by probe molecules affixed to the array, of target molecules, whether by sequence-mediated binding affinities, binding affinities based on conformational or topological properties of probe and target molecules, or binding affinities based on spatial distribution of electrical charge on the surfaces of target and probe molecules.
An xe2x80x9carrayxe2x80x9d, unless a contrary intention appears, includes any one, two or three dimensional arrangement of addressable regions bearing a particular chemical moiety to moieties (for example, biopolymers such as polynucleotide sequences) associated with that region immobilized on an array substrate. An array is xe2x80x9caddressablexe2x80x9d in that it has multiple regions of different moieties (for example, different polynucleotide sequences) such that a region (a xe2x80x9cfeaturexe2x80x9d or xe2x80x9cspotxe2x80x9d of the array) at a particular predetermined location (an xe2x80x9caddressxe2x80x9d) on the array substrate will detect a particular target or class of targets (although a feature may incidentally detect non-targets of that feature). Array features are typically, but need not be, separated by intervening spaces. In the case of an array, the xe2x80x9ctargetxe2x80x9d will be referenced as a moiety in a mobile phase (typically fluid), to be detected by probes (xe2x80x9ctarget probesxe2x80x9d) which are bound to the substrate at the various regions. However, either of the xe2x80x9ctargetxe2x80x9d or xe2x80x9ctarget probesxe2x80x9d may be the one which is to be evaluated by the other (thus, either one could be an unknown mixture of polynucleotides to be evaluated by binding with the other). An xe2x80x9carray layoutxe2x80x9d refers collectively to one or more characteristics of the features, such as feature positioning, one or more feature dimensions, and the chemical moiety or mixture of moieties at a given feature. xe2x80x9cHybridizingxe2x80x9d and xe2x80x9cbindingxe2x80x9d, with respect to polynucleotides, are used interchangeably.
Any given array substrate may carry one, two, four or more collections of array features disposed on a front surface of the array substrate. Depending upon the use, any or all of the collections of array features may be the same or different from one another and each may contain multiple spots or features. A typical array may contain more than ten, more than one hundred, more than one thousand more ten thousand features, or even more than one hundred thousand features, in an area of less than 20 cm2 or even less than 10 cm2. For example, features may have widths (that is, diameter, for a round spot) in the range from a 10 xcexcm to 1.0 cm. In other embodiments each feature may have a width in the range of 1.0 xcexcm to 1.0 mm, usually 5.0 xcexcm to 500 xcexcm, and more usually 10 xcexcm to 200 xcexcm. Non-round features may have area ranges equivalent to that of circular features with the foregoing width (diameter) ranges. At least some, or all, of the features may be of different compositions (for example, when any repeats of each feature composition are excluded the remaining features may account for at least 5%, 10%, or 20% of the total number of features). Interfeature areas will typically (but not essentially) be present which do not carry any polynucleotide (or other biopolymer of a type of which the features are composed). Such interfeature areas typically will be present where the arrays are formed by processes involving drop deposition of reagents but may not be present when, for example, photolithographic array fabrication processes are used. It will be appreciated though, that the interfeature areas, when present, could be of various sizes and configurations.
The array features can have widths (that is, diameter, for a round spot) in the range from a minimum of about 10 xcexcm to a maximum of about 1.0 cm. In embodiments where very small spot sizes or feature sizes are desired, material can be deposited according to the invention in small spots whose width is in the range about 1.0 xcexcm to 1.0 mm, usually about 5.0 xcexcm to 500 xcexcm, and more usually about 10 xcexcm to 200 xcexcm. Features which are not round may have areas equivalent to the area ranges of round features 16 resulting from the foregoing diameter ranges.
Each collection of array features may cover an area of less than 100 cm2, or even less than 50, 10 or 1 cm2. In many embodiments, the substrate carrying the one or more arrays will be shaped generally as a rectangular solid (although other shapes are possible), having a length of more than 4 mm and less than 1 m, usually more than 4 mm and less than 600 mm, more usually less than 400 mm; a width of more than 4 mm and less than 1 m, usually less than 500 mm and more usually less than 400 mm; and a thickness of more than 0.01 mm and less than 5.0 mm, usually more than 0.1 mm and less than 2 mm and more usually more than 0.2 and less than 1 mm. With arrays that are read by detecting fluorescence, the substrate may be of a material that emits low fluorescence upon illumination with the excitation light. Additionally in this situation, the substrate may be relatively transparent to reduce the absorption of the incident illuminating laser light and subsequent heating if the focused laser beam travels too slowly over a region. For example, substrate 10 may transmit at least 20%, or 50% (or even at least 70%, 90%, or 95%), of the illuminating light incident on the front as may be measured across the entire integrated spectrum of such illuminating light or alternatively at 532 nm or 633 nm.
Once the labeled target molecule has been hybridized to the probe on the surface, the array may be scanned by an appropriate technique, such as by optical scanning in cases where the labeling molecule is a fluorophore or by radiometric scanning in cases where the signal is generated through a radioactive decay of labeled target. In the case of optical scanning, more than one fluorophore can be excited, with each different wavelength at which an array is scanned producing a different signal. In optical scanning, it is common to describe the signals produced by scanning in terms of the colors of the wavelengths of light employed for the scan. For example, a red signal is produced by scanning the array with light having a wavelength corresponding to that of visible red light.
Scanning of a feature by an optical scanning device or radiometric scanning device generally produces a scanned image comprising a rectilinear grid of pixels, with each pixel having a corresponding signal intensity. These signal intensities are processed by an array-data-processing program that analyzes data scanned from an array to produce experimental or diagnostic results which are stored in a computer-readable medium, transferred to an intercommunicating entity via electronic signals, printed in a human-readable format, or otherwise made available for further use. Biopolymer/molecular array experiments can indicate precise gene-expression responses of organisms to drugs, other chemical and biological substances, environmental factors, and other effects. Molecular array experiments can also be used to diagnose disease, for gene sequencing, and for analytical chemistry. Processing of molecular array data can produce detailed chemical and biological analyses, disease diagnoses, and other information that can be stored in a computer-readable medium, transferred to an intercommunicating entity via electronic signals, printed in a human-readable format, or otherwise made available for further use.
FIG. 8 illustrates components of a molecular array scanner. Lasers 800a-b emit coherent light that passes through electro-optic modulators (xe2x80x9cEOMsxe2x80x9d) 810a-b with attached polarizers 820a-b. Each EOM and corresponding polarizer together act as a variable optical attenuator. A control signal in the form of a variable voltage is applied to each EOM 810a-b by controller 880. The controller 880 may include a suitably programmed processor, logic circuit, firmware, or a combination of software programs, logic circuits, and firmware. The control signal changes the polarization of the laser light, which alters the intensity of the light that passes through the EOM. In general, laser 800a provides coherent light of a different wavelength than that provided by laser 800b. For example, one laser may provide red light and the other laser may provide green light. The beams may be combined along a path toward a stage 800 by the use of full mirror 851 and dichroic mirror 853. The light from the lasers 800a-b is then transmitted through a dichroic beam splitter 854, reflected off fully reflecting mirror 856, and then focused, using optical components in beam focuser 860, onto a molecular array mounted on a holder 800. Fluorescent light, emitted at two different wavelengths (for example, green light and red light) from features of the molecular array in response to illumination by the laser light, is imaged using the optics in the focuser/scanner 860, and is reflected off mirrors 856 and 854. The two different wavelengths are further separated by a dichroic mirror 858 and are passed to photodetectors 850a-b. More optical components (not shown in FIG. 8) may be used between the dichroic mirror and the photodetectors 850a-b, such as lenses, pinholes, filters, and fibers. The photodetectors 850a-b may be of various different types, including photo-multiplier tubes, charge-coupled devices, and avalanche photodiodes.
A scan system causes a light spot from each laser 800a-b to be moved in a regular pattern about the surface of the molecular array. The molecular array is mounted to a stage that can be moved in horizontal and vertical directions to position light from the lasers onto a particular region at the surface of the molecular array, from which region fluorescent emission is passed back to the photodetectors via the optical path described above. An autofocus detector 870 is provided to sense and correct any offset between different regions of the molecular array and the focal plane of the system during scanning. An autofocus system includes detector 870, processor 880, and a motorized adjuster to move the stage in the direction of arrow 896.
The controller 880 receives signals from photodetectors 850a-b, called xe2x80x9cchannels,xe2x80x9d corresponding to the intensity of the green and red fluorescent light emitted by probe labels excited by the laser light. The controller 880 also receives a signal from autofocus offset detector 870 in order to control stage adjustment, provides the control signal to the EOMs 810a-b, and controls the scan system. Controller 880 may also analyze, store, and output data relating to emitted signals received from detectors 850a-b. 
The dynamic autofocus mechanism, described above, provides a means for maintaining a constant focus distance during scanning of a molecular array. However, any of various different focus distances may be selected to be held constant during a scan. In general, the focus distance must correspond to the depth of focus of the molecular array optics, but, in general, there is one or a small range of optimal focus distances for a particular molecular array scanner.
One parameter that can impact the optimal focus distance is thickness of an array substrate, e.g., glass thickness. The reason that different thicknesses of glass have different optimal focus positions is that a scanner looks xe2x80x9cthroughxe2x80x9d the glass. That is, the excitation light passes through a first surface, e.g., the front surface, of the glass and then through an opposing second surface, e.g., the back surface, of the glass where it then excites the molecules on the far side of the second, opposing surface. Even a scanner that detects features on the near/front surface of the glass may require different focus positions if the physical substrate moves closer or further away from the focusing lens as the glass thickness varies.
Designers, manufacturers, and users of molecular arrays have recognized a need for an automated focus-distance optimization method, where the method takes into account the thickness of the biopolymer array substrate, e.g., glass thickness, being scanned.
Relevant Literature
U.S. Pat. Nos. of interest include: 5,091,652; 5,260,578; 5,296,700; 5,324,633; 5,585,639; 5,760,951; 5,763,870; 6,084, 991; 6,130,745; 6,222,664; 6,284,465; 6,329,196; 6,371,370 and 6,406,849.
The present invention provides an automated method for determining an optimal focus distance for scanning a molecular array by a molecular array scanner, where the method is biopolymer array substrate thickness dependent in that the focus distance determined by the method is determined based in part on the thickness of the array substrate. Blocks of rows of a reference array are automatically scanned over a range of focus distances, or z-positions, to produce data providing a functional relationship between focus distance, or z-position, and measured signal intensities. The data is then processed by an array substrate thickness dependent routine that selects an optimal focus distance for data scans based on array substrate thickness. In calibrating a scanner according to the present invention, two or more reference substrates, e.g., reference arrays or reference members/elements, of different thickness are scanned according to the above protocol to identify an optimal focus distance for each different substrate thickness. Finally a monotonic curve of optimal focus distance versus array substrate thickness, and in most cases a linear fit to the curve, is obtained with the set of optimal focus distances obtained from the set of reference arrays. The coefficients of the fitted curve (slope and intercept for a linear fit) are stored as calibration data in the optical scanner to be used to find the optimal focal distance for each array substrate before the beginning of every scan using that scanner.
The present invention further provides a computer program product for use with an apparatus such as described herein. The program product includes a computer readable storage medium having a computer program stored thereon and which, when loaded into a programmable processor, provides instructions to the processor of that apparatus such that it will execute the procedures required of it to perform a method of the present invention.
The present invention also provides scanners calibrated according to the subject methods, as well as methods of using the subject calibrated scanners, e.g., in genomic and proteomic array based applications, and kits for use in calibrating optical scanners.